Dental caries is an infectious and transmissible disease and the mutans group of streptococci are the major causative agent. Dental caries still affects a large segment of the population, both in the United States and world-wide. Some strains of Streptococcus mutans produce bacteriocin-like substances (BLS) called mutacin which kill other bacteria of the same or closely related species. The production of mutacin is one characteristic of S. mutans which may contribute to its ability to colonize and hence, to cause disease. Because of their unique inhibitory properties, knowledge about mutacin may be applied to the eventual control of the pathogenesis of S. mutans. Unfortunately, very little is known about how mutacin is regulated or under what conditions it is made. Because these substances are made in small quantities and only under certain defined conditions, attempts to isolate them biochemically have been unsuccessful. For these and other reasons, a molecular genetic approach shows promise for determining not only the biochemical composition of mutacin, but also the genetic determinants responsible for regulation and production of this substance. Accordingly, this proposal entails the use of a powerful genetic technique known as insertional mutagenesis. Using this method, the genes responsible for mutacin production in S. mutans can be inactivated so that mutants unable to make this inhibitor can be identified. The method proposed for accomplishing gene inactivation involves the use of the transposon Tn9l6. This "jumping element" is capable of entering S. mutans and inserting itself in a random fashion within the chromosome; insertion into one of the mutacin genes will cause its inactivation. Once an inactivated mutant is found, the Tn9l6 and the DNA surrounding it (including the mutacin gene) can be transformed to E. coli where the Tn9l6 is unstable and often excises from the inactivated gene in a precise manner, thus reconstituting the original gene to its active state. Re-isolation of the gene from E. coli allows for its characterization in other bacteria, including S-mutans and S. sanguis. There are several long-term health-related interests for a study of mutacin production in S. mutans. First, once the gene which encodes the BLS is isolated, a means could be devised to enhance its production to the extent that larger quantities may be available for therapeutic application. Second, genetic manipulations could be performed which would enable a competing, non-cariogenic organism to produce mutacin against S. mutans. This constitutes what is called replacement therapy. Third, knowledge of the control or regulation of this gene will give us insight into one of the virulence factors of this important organism.